The E3 ubiquitin ligase \(CRL4^{Cdt2}\) targets proteins for destruction during DNA replication and following DNA damage (Havens and Walter, 2011). Its substrates contain "PIP degrons" that mediate substrate binding to the processivity factor PCNA at replication forks and damage sites. The resulting PCNA-PIP degron complex forms a docking site for \(CRL4^{Cdt2}\), which ubiquitylates the substrate on chromatin. Several \(CRL4^{Cdt2}\) substrates are known, including Cdt1, multiple CDK inhibitors, Drosophila E2f1, human Set8, S. pombe Spd1, and C. elegans \(Pol\eta\) (Havens and Walter, 2011). An emerging theme is that \(CRL4^{Cdt2}\) targets proteins whose presence in S phase is toxic. Here, I used Xenopus egg extract to characterize a new \(CRL4^{Cdt2}\) substrate, thymine DNA glycosylase (TDG). TDG is a base excision repair protein that targets G-U and G-T mispairs, which arise from cytosine and 5-methylcytosine deamination (Cortazar et al., 2007). Thus, TDG may function in epigenetic gene regulation via DNA demethylation, in addition to its canonical DNA repair function. A yet unknown E3 ubiquitin ligase triggers TDG destruction during S phase (Hardeland et al., 2007). Understanding TDG proteolysis in S phase is relevant to the regulation of DNA replication, DNA repair, and epigenetic control of gene expression. I discovered that TDG contains a variant of the "PIP degron" consensus and that TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and degron-specific manner during DNA repair and DNA replication in Xenopus egg extract. I further characterized what features of TDG contribute to its proteolysis. Interestingly, I could not identify any defects during DNA replication or during Xenopus embryonic development in response to a non-degradable form of TDG. Additionally, I examined how interactions between \(CRL4^{Cdt2}\) and multiple subunits of the PCNA homotrimer contribute to \(CRL4^{Cdt2}\) function. In a popular model, PCNA functions as a "tool belt" on DNA, binding three separate proteins through its individual subunits to facilitate rapid exchange of DNA replication and repair proteins as they are needed on DNA. To address this model, I generated a single chain polypeptide with three PCNA subunits connected through flexible linker sequences. I used this tool to determine how multiple PCNA subunits contribute to \(CRL4^{Cdt2}\) function. I found that a single wildtype subunit is sufficient for modest destruction of the \(CRL4^{Cdt2}\) substrate Cdt1, but complete Cdt1 destruction requires two separate wildtype subunits. Additionally, a single subunit was sufficient for leading strand elongation, challenging the "tool belt" model during DNA replication. I also discuss implications and future use of the single-chain PCNA.

Production, egg quality, behaviour, and physical condition were compared from Wk 20 to Wk 50 among three beak- trimmed commercial laying strains, Lohmann White (LW), H & N White (HN), Lohmann Brown (LB), and a non-commercial Cross between Rhode Island Red (male) and Barred Plymouth Rock (female) in conventional cages and in floor pens. All chicks were reared in their respective environments, and 450 and 432 pullets were housed at 18 and 7 weeks of age in cages and floor pens respectively. Hens in cages were provided with 688 cm2/bird and those in pens with over 6,000 cm2/bird, both of which are more than provided by commercial standards. Body weights and eggshell weights were higher for birds in floor pens than those in cages, and although they increased with age, body weight of hens in cages decreased at Wk 50. White-egg layers (LW, HN) used perches and nest boxes more than Brown-egg layers (LB, Cross). During the laying period, mortality was higher for all strains in cages and during the rearing period mortality was higher in floor pens for LB hens but not other strains. No aggressive behaviours were found, but the frequency of gentle feather pecking and pecking at the enclosure was higher in cages than in floor pens. Feather condition deteriorated over time in cages mainly because of contact with the cage wires whereas in floor pens, feather condition of birds at Wk 20 was not different from that at Wk 50. The frequency of keel bone deformities was higher for White-egg layers than for Brown-egg layers in cages and was higher for Cross hens than other strains in floor pens. Claws were longer in cages than in the floor pens. Foot condition was worse in floor pens than in cages. The welfare indicators used in this study showed that cages restricted the hens' behaviour compared to floor pens and resulted in higher laying period mortality, reduced body weight and deteriorated feather condition than floor pens. Both systems had advantages and disadvantages in regard to the hens’ health and welfare. The use of environmental complexities was strain specific in floor pens. The environment by genotype interactions suggests that the strain should be considered when considering alternative housing systems.

Two trials compared the effects of canola meal (CM) and juncea meal (JM) with and without dietary enzyme supplementation, on production performance, egg quality, bone quality and liver health characteristics of white- (WSLH) and brown-shell egg laying hens (BSLH). A total of 360 Lohmann LSL-Lite White (Trial 1, WSLH) and 300 Lohmann Brown-Lite (Trial 2, BSLH) laying hens were fed one of 10 isoenergetic and isonitrogenous diets (Soybean meal, 10 % CM, 20 % CM, 10 % JM or 20 % JM with or without a dietary enzyme cocktail of Superzyme OM and Bio-PhytaseTM) for 48 weeks. Based on the results of production performance, incidence of mortality, egg quality, bone quality, and liver health data, up to 20 % CM or JM can be included in diets of WSLH and BSLH without detrimental effects. Enzyme should be included in diets for both WSLH and BSLH.

This study investigates the etiology, diagnosis, and treatment of follicular stasis in female veiled chameleons (Chamaeleo calyptratus). Reproductive status was assessed by enzyme immunoassay of fecal metabolites of estrogen, progesterone, testosterone, and corticosterone; ultrasonography; and male introduction trials. Ultrasonography and hormone pattern analysis confirmed follicular stasis, while female response to male presence was inconclusive. Hormone patterns of corticosterone metabolites indicated a cyclical pattern consistent with reproductive events, but there was insufficient data to compare peak levels between ovulatory and non-ovulatory cycles. Ovulation induction was unsuccessful using either chicken GnRH-II, or a combination of progesterone and prostaglandin F2α. Feed restriction induced weight loss, but this was not directly related to changes in follicle size. Prevention of follicular development (i.e. contraception) was attempted using Depo-Provera and Lupron Depot, but neither treatment was effective. The outcomes of this study supplement the information on follicular stasis in reptiles, but further research is still needed. / NSERC, Toronto Zoo

Incorporation of full-fat flaxseed, and to a lesser extent, canola seed in diets to produce n-3-enriched products has attracted interest in the poultry industry. However, high amounts of nonstarch polysaccharides (NSP) in oilseeds compromise their nutritive value. The objectives of the current research were to develop enzyme supplements effective in cell wall depolymerization and viscosity reduction, particularly in flaxseed; to evaluate the effects of enzyme addition and feed processing on oil utilization and egg n-3 fatty acid deposition in broiler chickens and laying hens fed oilseed-containing diets; to characterize the NSP hydrolysis products and to investigate the effects of diet type and enzyme addition on growth performance and the incidence of necrotic enteritis (NE) in broiler chickens challenged with Clostridium perfringens. Results showed that diets containing high levels of flaxseed reduced egg production and shell quality in laying hens, and impaired final body weight and feed conversion ratio (FCR) in broiler chickens. Reducing flaxseed particle size via grinding did not improve the growth performance of broiler chickens, whereas diet pelleting showed more pronounced and beneficial effects in improving the nutritive value of flaxseed, particularly when intact seeds were used. Multicarbohydrase supplementation resulted in a significant depolymerization of cell wall polysaccharides in soybean, canola and flaxseed meals, which was followed by the production of water-soluble NSP hydrolysis products, and the reduction of flax mucilage viscosity in vitro was also evident. Enzyme addition to flaxseed-containing diets improved FCR of broiler chickens and egg production performance of laying hens, and facilitated egg n-3 fatty acid deposition. The C. perfringens challenge caused intestinal NE lesions and increased the mortality of broiler chickens with the highest NE mortality and intestinal C. perfringens counts observed in those fed flaxseed-containing diets. Enzyme supplementation to diets containing high levels of water-soluble NSP (wheat/barley- or wheat/barley/flaxseed-based) facilitated post-disease compensatory growth in pathogen challenged birds. This was accompanied by a numerical reduction of intestinal C. perfringens by 1.4 log10 cfu/g in birds fed the flaxseed-containing diets. Such findings indicated that enzyme addition may be used as a nutritional strategy to reduce the risk of NE development in broiler chickens.

The proximate causes triggering nest abandonment are unclear for most species, including the Yellow Warbler (Dendroica petechia), which abandons nests parasitized by cowbirds (via burial or desertion). Cowbird parasitism and rejection of parasitism are costly to some hosts; therefore cues affecting their responses have important evolutionary implications. Manipulative experiments showed that experimentally adding a cowbird egg elicited similar rejection frequencies (2008: 31.8%; 2009: 26.1%) as naturally laid eggs (2008: 27.1%; 2009: 20.0%). In 2008, interaction with an egg-removing model increased the probability of abandonment and the most aggressive individuals were more likely to bury the model cowbird egg. In 2009, eggs added to nests before sunrise were rejected at a frequency (29.7%) similar to eggs added to nests after sunrise (22.9%). Warblers returning to nests after egg addition peered significantly longer at their clutch than at control nests, shuffled their bodies more frequently when on the eggs and spent more time probing eggs with their bill once settled on their parasitized clutch. Furthermore, although non-mimetic blue eggs were not abandoned significantly more frequently than cowbird eggs (blue 31.1% versus cowbird 21.4%), only blue eggs were ejected from some nests. Thus, warblers use both tactile and visual cues to detect the presence of a parasitic egg in their nest. Eggs added to nests were not rejected at a lower frequency than naturally parasitized nests, as was recorded in a previous study. It is difficult to know whether this increase in abandonment of experimental eggs is due to phenotypic plasticity, genetic changes, or other factors. Egg recognition abilities may have changed because I have shown that the warblers’ behaviour changes before versus after egg addition, whereas no changes were recorded in an earlier study. Finally, not all individuals that buried eggs for the first time in 2009 (21.4%) buried again after being re-parasitized (5.3%), when less time remained in the breeding season relative to the first parasitism event. This suggests that egg rejection and host responsiveness in warblers, and likely other avian hosts that use abandonment as a form of rejection, is affected by environmental cues which may act as genetic expressers.

The song thrush (Turdus philomelos) was introduced to New Zealand from Britain during the mid 19th century and has become one of the most common terrestrial bird species in New Zealand. In this study, I surveyed a range of life-history traits in New Zealand song thrushes for comparison with traits of British thrushes. Clutch size, egg size and nest size have decreased, while the nestling period is shorter and the incubation period longer. This combination of changes suggests birds are investing less energy into each reproductive bout. Birds also appear unable to raise large broods, as nestling starvation is common in New Zealand, which suggests that food is limiting. I experimentally tested the ability of song thrushes to incubate enlarged clutches and broods, but productivity was not higher for enlarged broods and natural 3- and 4-egg clutches produced similar numbers of fledglings. Thus reduced clutch size may be an adaptation to the local environment. Differences in female incubation behaviour, with 3- and 4-egg clutches receiving higher levels of incubation and more visits per hour than 5-egg clutches, also suggest New Zealand thrushes have difficulty coping with clutches as large as those in Britain. The decrease in clutch size between New Zealand and Britain is in the direction and magnitude expected based on the change in latitude, which supports the hypothesis that factors affecting foraging time and food availability, such as daylength, temperature and rainfall, may be selecting for smaller clutches. Egg size was also found to have decreased in New Zealand, though this may be the result of smaller adult size. Hatchling mass was related to egg volume, but I found no effect of egg volume or clutch size on hatching success. However, nests containing more pointed eggs (i.e., abnormally-shaped eggs), had lower survival and hatching/fledgling success. Data from the national nest record database and my study both suggest that differences in song thrush productivity are the result of differential survival of nestlings. Nestling mortality due to starvation was common at Kowhai Bush, but rare in Britain, so either adult condition or food availability may be lowering reproductive success in New Zealand. High rates of nest failure (>65%) could also affect clutch size, but the strong directional selection imposed by food limitation during the nestling period suggests that increases in food supply would result in increased reproductive success even with the same levels of nest failure. When comparing clutch size throughout New Zealand, I found a significant, positive relationship with rainfall, which further suggests that food limitation may be the main factor driving changes in life-history traits of song thrushes in New Zealand.

The objective of this thesis was to evaluate inclusion of up to 35% by-product DDGS (with reduced Ca and P) with and without addition of a proprietary premix (enzyme and antioxidant; Alltech Inc.) on productive performance and egg quality of white and brown egg laying hens. A total of 288 white or brown hens were randomly allocated to one of the following treatments: 1) corn-soybean meal (control), 2) 25% DDGS, 3) 25% DDGS plus premix, 4) 35% DDGS, and 5) 35% DDGS plus premix. Hen body weight values were impaired with addition of 25 and 35% DDGS when compared to the control. Premix helped maintain body weight comparable to control in the brown hens; however was not noted in white hens. In the second dietary phase, addition of DDGS increased feed intake in white egg laying hens and was maintained with the premix. Brown hens on premix came into lay faster than the control and DDGS diets, but DDGS reduced overall egg production. Haugh units, yolk color, and purchase intent scores were increased in brown eggs with DDGS diets. White eggs had increased yolk color with DDGS. In conclusion, up to 35% DDGS is acceptable in white laying hen diets.

The object of this research was to determine if an acceptable angel food cake alternative could be produced that had reduced calories and sucrose content. This was accomplished through replacing sucrose in meringue, angel food cake batter, and baked angel food cakes with polydextrose and either sucralose, acesulfame-K, or Rebaudioside A at different replacement levels (25, 50, 75, 100%). Meringue and cake batter properties were measured using rheological techniques. Baked angel food cakes were analyzed based on height, weight loss, moisture content, color, and TPA analysis. With meringue batter, 100% sucrose replacement was unacceptable since undissolved polydextrose made analyzing and end products impractical. While 75% sucralose and acesulfame-K sucrose replacement exhibited comparable air incorporation to the sucrose control in cake batter, baked angel food cakes showed a decrease in functional properties. Polydextrose was likely the cause. 25% sucralose and acesulfame-K sucrose replacement were no different from sucrose cakes in regards to height, overall textural appearance, crumb pore size, and hardness. These cakes resulted in an overall calorie reduction of 18.7%. In every experiment, Rebaudioside A replacement treatments exhibited trends opposite of sucralose and acesulfame-K treatments. Rebaudioside A treatments performed the worst for rheological properties and TPA analysis.

The proximate causes triggering nest abandonment are unclear for most species, including the Yellow Warbler (Dendroica petechia), which abandons nests parasitized by cowbirds (via burial or desertion). Cowbird parasitism and rejection of parasitism are costly to some hosts; therefore cues affecting their responses have important evolutionary implications. Manipulative experiments showed that experimentally adding a cowbird egg elicited similar rejection frequencies (2008: 31.8%; 2009: 26.1%) as naturally laid eggs (2008: 27.1%; 2009: 20.0%). In 2008, interaction with an egg-removing model increased the probability of abandonment and the most aggressive individuals were more likely to bury the model cowbird egg. In 2009, eggs added to nests before sunrise were rejected at a frequency (29.7%) similar to eggs added to nests after sunrise (22.9%). Warblers returning to nests after egg addition peered significantly longer at their clutch than at control nests, shuffled their bodies more frequently when on the eggs and spent more time probing eggs with their bill once settled on their parasitized clutch. Furthermore, although non-mimetic blue eggs were not abandoned significantly more frequently than cowbird eggs (blue 31.1% versus cowbird 21.4%), only blue eggs were ejected from some nests. Thus, warblers use both tactile and visual cues to detect the presence of a parasitic egg in their nest. Eggs added to nests were not rejected at a lower frequency than naturally parasitized nests, as was recorded in a previous study. It is difficult to know whether this increase in abandonment of experimental eggs is due to phenotypic plasticity, genetic changes, or other factors. Egg recognition abilities may have changed because I have shown that the warblers’ behaviour changes before versus after egg addition, whereas no changes were recorded in an earlier study. Finally, not all individuals that buried eggs for the first time in 2009 (21.4%) buried again after being re-parasitized (5.3%), when less time remained in the breeding season relative to the first parasitism event. This suggests that egg rejection and host responsiveness in warblers, and likely other avian hosts that use abandonment as a form of rejection, is affected by environmental cues which may act as genetic expressers.